As is well known, carnitine (.beta.-hydroxy-.gamma.-trimethyl-amino butyric acid) contains a center of asymmetry and therefore, carnitine exists in two stereoisomeric forms, the D and the L forms.
L-carnitine is normally present in the body where it functions to carry activated long-chain free fatty acids through the mitochondrial membrane. Since the mitochondrial membrane is impermeable to acyl CoA derivatives, long-chain free fatty acids can enter only when esterification with L-carnitine has taken place. The carrier function of L-carnitine is exerted both by transporting active long-chain fatty acids from the sites of their bio-synthesis, for the example the microsomes, to the mitochondria where they are oxidized, and by transporting acetyl CoA from the mitochondria, wherein it is formed, to the extramitochondrial sites where the synthesis of long-chain fatty acids occurs, e.g., in the microsomes wherein acetyl CoA can be utilized for synthesizing cholesterol and fatty acids.
While it has been established that the laevorotatory isomer (L-carnitine) exclusively is the biological form (D-carnitine has never been detected so far in mammalian tissues), the D,L-carnitine racemate has been used for a number of years for different indications. For example, D,L-carnitine is sold in Europe as an appetite stimulant, and it has been reported that the material has an effect on the growth rate of children; see e.g., Borniche et al., Clinica Chemica Acta, 5, 171-176, 1960 and Alexander et al., "Protides in the Biological Fluids", 6th Colloquim, Bruges, 1958, 306-310. U.S. Pat. No. 3,830,931 describes improvements in myocardial contractility and systolic rhythm in congestive heart failure which can often be obtained through administration of D,L-carnitine. U.S. Pat. No. 3,968,241 describes the use of D,L-carnitine in cardiac arrhythmias. U.S. Pat. No. 3,810,994 discloses the use of D,L-carnitine in the treatment of obesity.
Recently, however, there has been an increasing emphasis on the importance of utilizing exclusively the carnitine laevorotatory isomer for at least some therapeutic applications. It has, in fact, been shown that D-carnitine is a competitive inhibitor of carnitine-linked enzymes such as carnitine acetyl transferase (CAT) and carnitine palmityl transferase (PTC). Moreover, recent evidence suggests that D-carnitine can deplete the L-carnitine level of heart tissue. Consequently, it is essential that L-carnitine exclusively be administered to patients under medical treatment for heart diseases or lowering of blood lipids.
Several processes have been proposed for producing carnitine on an industrial scale. The chemical synthesis of carnitine unavoidably leads, however, to a racemic mixture of the D and L isomers. Consequently, resolution methods have to be employed to obtain the separate optical antipodes from the racemate.
A typical resolution method wherein D,L-carnitinamide hydrochloride is used as the starting compound for resolution is disclosed in Belgian Pat. No. 660039. Such process comprises the use of D-camphoric acid for producing the D-camphorate of D,L-carnitinamide. An alcoholic solution of this compound is subjected to fractional crystallization so as to give the L-isomer as the first fraction to precipitate from the solution.
In order to form the D-camphorate of D,L-carnitinamide, it is first necessary to form the ammonium salt of D-camphoric acid with ammonia; the ammonium D-camphorate that is formed is then converted to silver D-camphorate by the action of silver nitrate. Since the carnitinamide is in the hydrochloride salt form, the formation of this silver salt is essential in order to eliminate the chloride ion. Such a process is, therefore, very expensive (because of the imperative use of the silver compound) and difficulty to carry out industrially in that the various steps of the process have to be carried out away from the light in order to avoid marked blackening of the reaction vessels, due to the large quantity of AgCl which is formed. The D-camphorate of D,L-carnitinamide may, in addition, be rendered impure by the presence of silver ions.
Moreover, after the D-camphorate of L-carnitinamide has been crystallized out of the alcoholic solution, further steps are needed to eventually convert it into L-carnitine.